Nuclear Factor-kappa B is not lnvolved in Titanium Dioxide- Donald WiLsoN, Mazen ZAQouT, Jeong-Hoon HEo2, Eun-Kee PARK3, Chul-Ho OAK4 and

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1 J UOEH 34 (2): (2012) 183 [Short Report] Nuclear Factor-kappa B is not lnvolved in Titanium Dioxide- Induced lnflammation Donald WiLsoN, Mazen ZAQouT, Jeong-Hoon HEo2, Eun-Kee PARK3, Chul-Ho OAK4 and Susumu UENo l Department of Occupational Toxicology, lnstitute oflndustrial Ecological Sciences, University of Occupational and Environmental Health, Japan. Yahatanishi-ku, Kitakyushu , Japan 2 Department of Molecular Biolog: ソ (ft lmmunology, K sin 伽 vez 吻 Co ege ofmedicine, Busan , Korea 3Department ofmedical Humanities and Social Medicine, Kosin University College ofmedicine, Busan , Korea Department ofrespiratory Medicine, Kosin University, Korea, Kosin University College of Med た ine Busan 602_702 KorE~a s 一 vvu v-v v v r v J Abstract : Research over recent years have shown that titanium dioxide (TiO2) nanoparticles (NPs) induce inflammation in various lung, kidney, liver and brain cells. Although the mechanism of inflammation is unclear, existing literature suggests the underlying role of oxidative stress. On the other hand, it has also been shown that nuclear factor-kappa B (NF- KB) is activated in response to pro-inflammatory cytokines. ln this study we investigated the involvement of NF-KB in TiO2-induced inflammation in human lung adenocarcinomic epithelial cells (A549 cells). After 24h of treatment, IL-8 protein release from A549 cells, induced by 10, 50 and 250 ptg/ml of P25 TiO, NPs, were statistically significantly raised, compared to that ofthe control. This finding corroborates existing literature in that TiO2 NPs induce a dose-dependent increase in the release of IL-8 protein when exposed to A549 cells. However, the binding ofnf-kb DNA was not affected after 6 h of incubation with P25. Therefore, NF-KB DNA binding is not the likely transcription pathway that leads to TiO, 一 induced inflammation. Key words : Titanium dioxide nanoparticles, TiO,, inflammation, A549 cells, NF-i(B. (Received February 15, 2012, accepted April 27, 2012) Corresponding Author : Donald Wilson, mo, Phl), Department of Occupational Toxicology, lnstimte of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan. Yahatanishi-ku, Kitakyushu , Japan, Tel : ,Fax= , wilson@med.uoeh-u.ac.jp

2 184 D WiLsoN et al Introduction Titanium dioxide (TiO2), a noncombustible and odorless white powder, exists naturally in the forms of anatase, rutile and brookite. However, its more familiar form is as a white pigment for a wide range of paints, paper, plastics, ceramics, etc. TiO2 becomes transparent at the nanoscale (panicle size 100 nm), is able to absorb and reflect UV light, and has consequently been popularly used in sunscreen lotions and creams. ln addition, nano-sized TiO2 is nowadays produced abundantly and used widely because of its high stability and its anti-corrosive and photocatalytic propenies [1]. Due to the extensive use of TiO2, and the fact that nano-sized particles are generally more toxic than their larger-sized counterparts [2], concern for the potential risk of TiO2 nanopanicles (NPs) to humans has increased. Based on the recommendations of the lnternational Agency for Research on Cancer (IARC) [1], the U.S. National lnstitute for Occupational Safety and Health (NIOSH) recently deemed inhaled ultrafine or nano-sized TiO2 a potential occupational carcinogen, and has consequently recommended exposure limits to minimize the cancer risk from exposure to ultrafine TiO2 [3]. The NIOSH also suggested that the most plausible mechanism for TiO2 carcinogenesis is a nonspecific chemical interaction of the particle with the cells in the lung, characterized by persistent inflammation and mediated by secondary genotoxic processes [3]. TiO2 NPs have been shown to induce inflammation in exposed cell lines. A study by Singh et al (2007) demonstrated that only the nano-sized TiO2 particles [4], and not the slightly larger fine particles, were found to elicit oxidative stress and IL-8 release from A549 cells. ln addition, this TiO2-induced inflammation in A549 cells was proportional to the particle surface area that was exposed, suggesting that a large surface area alone in the lung may be sufficient to initiate inflammation by particles [5]. Although the mechanism of inflammagenicity induced by TiO2 NPs remains unclear, previous studies suggest that oxidative stress should be considered as an event that underlies and regulates cellular signaling that leads to inflammatory, proliferative, and genotoxic effects ofparticle substances [6]. Activation of nuclear factor-kappa B (NF-KB) has also been shown in human peripheral blood mononuclear cells, airway epithelial cells and lung tissue in response to pro-inflammatory cytokines [7]. This research group also suggested that activation of NF-ic13 occurs through the generation of reactive oxygen species (ROS). We wanted to investigated in this study whether the TiO, NPs-induced inflammogenicity is caused by oxidative-stress-driven DNA damage, via overexpression of NF-KB in human lung epithelial cells. Materials and methods Particle preparation and charac 彪 Tization P25 TiO2 nanoparticles (BET specific surface area of 53.8 m2/g) were obtained from Degussa. Crystalline silica (Min-U-Sil 5) quartz (BET specific surface area of 5 m2/g) was used as a positive control. The panicles were suspended as follows: 1.5 grams of P25 TiO2 powder was suspended in 100 ml of distilled water, and sonicated for 15 minutes by a Branson Digital

3 (承)おρヨ自書モNF-KB is not lnvolved in Titanium Dioxide-lnduced lnflammation 185 Cell Disruptor Sonifier 250; this process was repeated 3 times. The particle suspension was then centrifuged at 3,000 g for 20 minutes, at 200C. The supernatant was carefully collected and filtered through a 1 pm filter to remove the large agglomerates ( 1 pm). A given volume of particle suspension was evaporated, after which the weight of the remaining evaporate was measured and the P25 TiO2 concentration determined (w/v; in mg/ml). lmmediately before application to cells, the particle suspension was mixed with double-strength (2 ) serum free Dulbecco s Modified Eagle Medium (DMEM) and sonicated for 15 minutes in a water bath. The hydrodynamic size distribution by number of P25 TiO2 particles suspended in water was analyzed using a DLS Zetasizer Nano (Malvern lnstruments, U.K.), and the average particle size was calculated to be ± 5.14 nm (Fig. 1). Because the particles that were ultimately treated with the A549 cells were suspended in serum 丘 ee DMEM(SFM), we also tried to measure the hydrodynamic particle size of particles suspended in SFM. Unfb 血 lnately, because the suspensions were unstable, the results were inconclusive and therefore not used 一 M - 0工 ゾ Hydrodynamic Particle Size (nm) [log scale] Fig. 1. Dynamic Light Scattering (DLS) spectra of the P25 titanium dioxide (TiO2) nanoparticles (NPs) in water. The graph shows the logarithmic scale of the hydrodynamic particle size (nm) against the particle number (O/o). TiO2 NPs were dispersed in ultrapure water, and sonicated 3 times for 15 minutes. After centrifugation, the supernatant was carefully collected and filtered. The hydrodynamic particle number and size distribution in water suspensions was analyzed for 3 samples of the same TiO2 concentration, using a DLS Zetasizer Nano (Malvern lnstruments, U.K.). The three peaks on the graph are representative of the 3 samples, the average particle size ofwhich was calculated to be ± 5.14 nni (represented by the dotted vertical line).

4 186 D WiLsoN et al Cell culture and treatment Human long adenocarcinomic alveolar basal epithelial cells(a549 cells), were obtained 丘 om Keio University, Tokyo. The cells were maintained in DMEM (lnvitrogen, Carlsbad, CA), supplemented with 100/o fetal bovine serum (Hyclone, Logan UT), and 100 U/ml penicillin/streptomycin (lnvitrogen) at 370C and 50/o CO2. The cells were plated in 6-well plates at a density of cells /well. For the assays, the cells were treated for 6, 12 or 24 hours, with P25 TiO2 NPs at final concentrations of 2, 10, 50 and 250 pg/ml, and with Min-U-Sil explained in p 184 at final concentrations of 48, 96, 192 and 384 pg/ml. The cells were cultured to 800/o confluency in DMEM with 100/o fetal bovine serum (FBS) and then starved for 24 hours to arrest their growth before treatment. Measurement ofll-8 by ELISA Human IL-8 released from the cells was measured by Human IL-8 ELISA kit (Thermo Scientific, Rockford IL). Following incubation with the NPs for 24 hours, cell supernatants were recovered and centrifuged at 13,000 x g for 10 minutes and the particle-free cell supernatants were stored at 一 800C until analysis. ELISA was performed as described in the manufacturer s instructions. Briefly, 50 pl of standard diluents or cell supernatants were added to an anti-human IL-8 pre-coated 96-well strip plate and incubated for 1 hour at room temperature. The plate was washed 3 times with Wash Buffer, and 50 pl of the biotinylated antibody reagent was added to each well and incubated for 1 hour at room temperature. After washing 3 times with Wash Buffer, 100 pl of streptavidin-hrp solution was added to each well and incubated for 30 minutes at room temperature. After washing 3 times with Wash Buffer, 100 pl of 3,3 C5,5 一 Tetramethylbenzidine (TMB) solution was added to the well and incubated in a dark room for 30 minutes at room temperature. The reaction was stopped by adding 100 pl of Stop Solution. Absorbance was measured on an ELISA plate reader at 450 and 550 nm. Human IL-8 amounts were determined using the standard curve and expressed as pg/ml. NF-KB determination 1. Nuclei extraction After exposure of the cells to NPs, they were washed 3 times in ice-cold phosphate-buffered saline (PBS). The cells were scraped in PBS and then pelleted for 15 seconds at 14,000 g. The cells were re-suspended in 400 pl of lysis buffer (10 mm 4 一 (2-hydroxyethyl) 一 1-piperazineethanesulfonic acid (HEPES), ph 7.8, 50 mm KCI, 2 mm MgCl,, l mm dithiothreitol (DTT), O.1 mm ethylenediaminetetraacetic acid (EDTA), O.4 mm phenylmethanesulfonylfluoride (PMSF), O.2 mm NaE O.2 mm sodium Orthovanadate, l pg/ml Leapeptin) and incubated for 15 minutes on ice. 100/o NP-40 were added to the mixture and then centrifuged at 14,000 g for 30 seconds. The supematant containing the cytoplasmic 丘 action was aspirated and retained. The pelleted nuclei were re-suspended in 50 pl of extract, and mixed for 20 minutes by rotation, then centrifuged for 5 minutes at 14,000 g. The supernatant containing nuclear proteins was collected and saved for further Western blotting (WB) and electrophoretic mobility shift assay (EMSA). Protein concentrations were measured (by Bradford method) prior to saving the supernatant.

5 桝盤 げ O 巴 f 熱翫 窮饗激 暁:1,卸 昌,二勧, 逮 冒 い!L げi灘 ゴ汐 麟亭 ミリヒ リ 判 に 蔑擁ミリ _ボ 艇l焦f}Oそ, 暫局ノ. 閏.弼:黛鱒喉F カ ド.,ゴゴビF ろ廊亭聯!!菊勢1弼守讃昏 罰,,も X 野が し競 D ダ 賦 げら~しT.悟, LNF-KB is not lnvolved in Titanium Dioxide-Induced lnflammation Electrophoretic mobility shift assay (EMSA) The NF-ic13 was determined by EMSA Kit (E33075 from Molecular Probes lnvitrogen Detection Technologies), according to the manufacturer s instructions. Briefly, DNA-protein binding assays were carried out with the nuclear extract. Synthetic complementary NF-i(B (5 一 AGT TGA GGG GAC TTT CCC AGG C-3 j binding oligonucletides (Bioneer) were 3 一 biotinylated using the biotin 3 一 end DNA labeling Kit (Pierce) according to the manufacturer s instructions, and annealed for 30 minutes at room temperature. The reaction mixture was electrophoretically separated on a 40/o polyacrylamide gel in O.5X Tris-borate buiifer and transferred to a nylon membrane. The transferred DNAs were cross-linked to the membrane at 120 mj/cm2. Horseradish peroxidase-conjugated streptavidin was use according to the manufacturer s instructions to detect the transferred DNA. Results 1L-8 releaseプれ )m/4549 cells bソ乃 02ハJPs When A549 cells were exposed to TiO2 NPs for 24 hours, the concentrations of IL-8 released in the culture medium were increased in a TiO2-dose dependent manner (Fig. 2). The increase of IL-8 release induced by 10, 50 and 250 ptg/ml of TiO2 was statistically significant, compared to that ofthe control sample. *** ( 一 _bdω)oqi]一 o ヒ,ヂb 輿窺 鰍 F 雷 f 5 無 w, 一 ** *鯖一 1:髪..P,} 玲.,潔 F 〆瀧爆墾 ヒ脇ヒ ヒ 磯ヘクタール コ 哩トン 帆 ぺ { 憾そ }1撤羅@ 1 戸 繍, トン詞磁臨 が蘭弼 C瀦難 E 戸 ρハ ーセント d 霧縷 み〆ノ硲難ド鋼鷹ト カ. 罫捗 m ぬ 塑二@! イ.P 腐 w. 賜 咋 f 勤トン 1 紙 ワット巨飢ゲキロげ 辱,雛1昌 μ }戸遷ュ: 1購鰍灘 曙ヘクタール@ 辱ルi 団 7 鵠 叫, 年 ~ 鳶ミ目鰭馳.〆,冠 ノ.ご }ニゴ ごl二 心 雛晦y 艇 y ワ霧 縞 ヤ叛 TiO2 concentration (pg/ml) Fig.2. 皿 一 8 protein release from A549 cells,24 hours after exposure to TiO, NPs. After the A549 cells were treated with and without TiO2 NPs for 24 hours, at concentrations of 2, 10, 50 and 250 ptgtml, the supernatant was collected and cenuifuged at 13,000 g for 10 min. The concentrations of released IL-8 in the culture media were measured using Enzyme-linked irnmuno sorbent assay (]ilisa) Kit. The amounts of I]L-8 are expressed as mean±sd. Asterisks on bar graphs indicate statistically sign 正 icant differences compared to the control (no TiO2), *: P O.05, * : P O.01 and ***: P O.OO 1 (one way ANOVA followed by the Dunnerfs test).

6 188 D WiLsoN et al EffeCt O. プTio 乙 Ps on NF 一 κb DNA binding acti 吻 The effect of TiO2 NPs exposure on NF-ic13 DNA binding was evaluated after 6 hours of exposure, when the expressions of NF-i(B bands were verified by EMSA. As shown in Fig. 3, TiO2 NPs did not affect the binding activity ofnf-kb DNA. TiO, (pg/ml) NC C PC Fig. 3. Effect of TiO2 NPs on NF-KB expression. After the A549 cells were incubated for 6 hours with and without TiO2 NPs, they were washed, scraped and pelleted, and the nuclear proteins were extracted. The extracted nuclear fraction was used for the determination of NF-KB by Electrophoretic Mobility Shift Assay (EMSA). This diagram is the representative EMSA gel at 6 hours of treatment with TiO2 NPs, on NF-i(B expression. The NC lane represents the negative control for EMSA, C the control for treatment (No treatment), and the positive control (PC). Discussion IL-8 is an inflammatory chemokine that functions mainly as a chemo-attractant for leukocytes. Existing literature reveals that measurement of IL-8 released from cultured cells is considered an indicator of inflammagenic potency of TiO2 NPs [4, 5]. ln this study, we showed that after 24 hours of exposure ofa549 cells to TiO2 NPs, the level of IL-8 released increased in a statistically significant, TiO2 dose-dependent manner in the culture medium. These results are consistent with those of Singh et al [4] and Monteiller et al [5], suggesting that TiO2 NPs can induce inflammation in exposed cell lines. We also found in this study that TiO2 NPs did not cause an alteration in the expression of NF-KB, a protein complex that controls the transcription of DNA, NF-ic13 is considered a first responder to hamm血 l cellular stimuli such as ROS[8], and the transcription ofil-8 is NF-K:B de 一

7 NF-KB is not lnvolved in Titanium Dioxide-lnduced lnflammation 189 pendent [9]. We investigated the effect of TiO, NPs on NF-i(B expression based on the fact that TiO2 NPs induce ROS production in A549 cells [4]. However, our negative findings suggest that NF-icB DNA binding is not the likely transcription pathway that leads to TiO2-induced inflammatlon. Alternative explanations for these results can be made by examining other transcriptional factors associated with IL-8 protein release. lt has been previously proposed that various motifs found on the 5-flanking region of the human IL-8 promoter have the potential to bind a number of important transcription factors [10]. Two of these factors, reported by Yong et al are Nuclear Factor lnterleukin-6 (NF-IL-6), Activator Protein-1 (AP-1) [11]. Nagarsekar et al proposed the contribution of heat shock activated transcription factors (HSFs) [12]. Yong et al found that bradykinin, a non-cytokine mediator, caused IL-8 release from human airway smooth muscle (HASM) cells, and they suggested that NF-i(B and other transcriptional factors, such as AP-1 and NF-IL-6, are likely to be involved in IL-8 release. Since IL-8 is also known to contain multiple heat-shock response elements (HSE) 一 like sequences, it is possible that IL-8 activation can also be mediated by HSFs [12, 13]. Conclusion In conclusion, it is unlikely that NF-KB is involved in the inflammagenic ability of TiO, NPs. However, our findings corroborate existing knowledge that TiO2 NPs cause cellular inflammation. ln view of our findings that NF-KB expression was not affected, and on the aforementioned findings of previous studies, our future plan is to investigate the involvement of other transcriptional factors, namely AP-1, NF-IL-6 and HSFs, in TiO2-induced IL-8 release. Acknowledgements This study was j ointly funded by a UOEH Research Grant for the Promotion of Occupational Health, No. H23( 平成 23 年度産業医学 産業保健重点研究費 ),and a UOEH Grant fbr Advanced Research, No. H21-H23( 平成 21 年度 ~23 年度産業医科大学高度研究費 ). References 1 O International Agency for Research on Cancer (IARC) (2006): Titanium dioxide Summary of data reported. [Online; cited January 7, 2012] Lam CW, James JT, McCluskey R & Hunter RL (2004): Pulmonary toxicity of single wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicol Sci 77: National lnstitute for Occupational Safety and Health (NIOSH) (2011): Occupational exposure to titanium dioxide. Current lntelligence Bulletin 63: 2-3 Available at url: cdc.gov/niosh/ docs/ /.pdf 4. Singh S, Shi T, Duffin R et al (2007): Endocytosis, oxidative stress and IL-8 expression in human

8 190 D WiLsoN et al lung epithelial cells upon treatment with fine and ultrafine TiO2: Role of the specific surface area and of surface methylation ofthe particles. Toxicology Appl Pharmacol 222: Monteiller C, Tran L, MacNee W, Faux S, Jones A, Miller B & Donaldson K (2005): The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area. Occup Environ Med 64: Donaldson K & Stone V (2003): Current Hypotheses on the Mechanisms oftoxicity of Ultrafine Particles. Ann Ist Super Sanita 39: Ralman 1, Mulier B, Gilmour P S, Watchorn T, Donaldson K, Jeffery PK & MacNee W (2001): Oxidant-mediated lung epithelial cell tolerance: the role of intracellular glutathione and nuclear factorkappab. Biochem Pharmacol 62: Perkins ND (2007): lntegrating cell-signalling pathways with NF-KB and IKK function. Nat Rev Mol Cell Biol 8: Ql/O11. Sen CK (2000): Cellular thiols and redox-regulated signal transduction. Curr Top Cell Regul 36: 1-30 Choi EY, Park ZY, Choi EJ, Oh HM, Lee S, Choi SC, Lee KM, lm SH, Chun JS & Jun CD (2007): Transcriptional regulation of IL-8 by iron chelator in human epithelial cells is independent from NFkappa B but involves ERKl/2-and p38 kinase-dependent activation ofap-1. J Cell Biochem l O2 二 Zhu YM, Bradbury DA, Pang L & Knox AJ (2003): Transcriptional regulation of interleukin (IL) 一 8 by bradykinin in human airway smooth muscle cells involves prostanoid-dependent activation of AP-1 and nuclear factor (NF) 一 IL-6 and prostanoid-independent activation of NF-kappa B. Biol Chem 278: Nagarsekar A, Hasday JD & Singh IS (2005): CXC chemokines: a new family of heat-shock proteins? Immunol lnvest 34: Singh IS, Gupta A, Nagarsekar A, Cooper Z, Manka C, Hester L, Benjamin IJ, He JR&Hasday 皿 ) (2008): Heat Shock Co-Activates lnterleukin-8 Transcription. Am J Respir Cell Mol Biol 39:

9 NF-KB is not lnvolved in Titanium Dioxide-Induced lnflammation 191 二酸化チタンナノ粒子が誘導する炎症反応に核内因子 ms が関与していない ドナルドウィルソン 1, マゼンザクート 1, ジョンフンホー 2, ユンキーパク 3, チュルホオーク 4, 上野晋 1 1 産業医科大学産業生態科学研究所職業性中毒学教室 2 韓国釜山市高神大学医学部分子生物学 免疫学部門 3 韓国釜山市高神大学医学部医療人文科学 社会医学部門 4 韓国釜山市高神大学医学部呼吸器内科部門 要旨 : 近年における研究によると, 二酸化チタン (Tio2) ナノ粒子が肺, 腎臓肝臓および脳など様々な細胞で炎症反応を誘導することが示されている. その炎症反応が誘導されるメカニズムについては未だ明らかとされていないが, これまでの文献によると酸化ストレスがその根本的な役割を果たしていることが示唆されている. 一方, 核内因子 mb(nf-kb) が炎症性サイトカインに反応して活性化されることも示されている. そこで本研究では, ヒト肺上皮腺癌 (A549) 細胞において, Tio2が誘導する炎症反応にNF-KBが関与しているかを検討した.24 時間処理後におけるA549 細胞からのIL-8 放出量は対照群と比較して,10,50および250μg/m1のP25 Tio2ナノ粒子において統計学的に有意に増加した.IL-8 放出量の結果は, 報告された知見を裏付けるものである. しかしながら,P25で6 時間処理した後のNF-kBのDNAへの結合性には影響を与えなかった. NF-KB 検討の結果から,NF-KBのDNAへの結合がTiO2 誘導性の炎症反応を生じるための転写経路とは考えにくい. キーワード = 二酸化チタンナノ粒子,TiO2, 炎症反応,A549 細胞, NF-mb. JUOEH( 産業医大誌 )34(2) (2012)